The Effect Of Protective Autophagy Induced By Melittin On HCC Cells Growth

BackgroundAutophagy is highly evolutionarily conserved process in virtually all eukaryotic cells,which is closely related to cell survival.Anticancer medicines which could target autophagy may become the new ones for therapeutic of cancer strategic treatment.It is an important drug development means to derive antitumor active ingredient from biological toxin for screening of natural medicine.This is also one of the ways of practice the theory of traditional Chinese medicine on cancer treatment.Melittin can inhibit the proliferation and induce apoptosis of hepatocellular carcinoma cells through multiple pathways.It was observed that melittin can induce the formation of autophagy in human hepatoma cell line and the over expression of autophagy marker protein Beclin 1,LC3-II by Transmission electron microscope,Confocal laser scanning microscope and Immunofluorescence.All the initially results confirmed that autophagy is also one of the tumor suppressor multi-targets of melittin.A systematic and depth study on melittin anti-liver cancer function was made in our department for many years.Thus,in this project,we intend to observe melittin-induced autophagy in human hepatoma cell line HepG2,SMMC-7721and analyse the mechanism of autophagy in cell death,and we also focus on the autophagy-related signaling pathway to explore targets melittin in human hepatoma carcinoma HepG2,SMMC-7721 autophagic cell death.All the above experimental researches will be the basis of the development and utilization of melittin.ObjectiveThis issue studies human hepatoma cell line HepG2,SMMC-7721 and constructs human hepatocellular carcinoma cell bearing nude mice model on this basis.We investigate the mechanism of melittin on liver cancer cells mainly from the cell proliferation,autophagy and apoptosis by activating and inhibiting autophagy respectively.It contributes to understand the role of autophagy in melittin inducing human hepatoma cell death clearly.We investigate the possible mechanism of the effect of melittin inducing human hepatoma cell death by testing autophagic and apoptotic protein pathways to provide the experimental basis and new ideas for study on melittin anti-liver cancer function.MethodsExperiment 1:Melittin inhibits HCC cells proliferation and induces autophagy.1、MTT was used to detect the proliferation inhibition of hepatoma HepG2,SMMC-7721 cells in different concentration of Mel for 24 h,48 h,72 h respectively.Inverted phase contrast microscope was applied to observe the morphological change of hepatoma HepG2,SMMC-7721 cells in different concentration of Mel for 24 h.Annexin V-FIFC/PI double staining was used to detect the apoptosis rate of hepatoma HepG2,SMMC-7721 cells in different concentration of Mel for 24h.Trypan blue staining was used to detect the cell death of hepatoma HepG2,SMMC-7721 cells in different concentration of Mel for 24h.2、Western Blot was used to detect the expression of LC3Ⅰ/Ⅱ,Beclin 1 and p62 of hepatoma HepG2,SMMC-7721 cells in different concentration of Mel for 24 h and Mel 5μg/mL for different time respectively.Transmission Electron Microscope was applied to observe the apoptotic and autophagic change of hepatoma HepG2,SMMC-7721 cells in Mel 5μg/mL for 0h,24h.Immunofluorescence was used to assess expression of Beclin 1 of hepatoma HepG2,SMMC-7721 cells in Mel.Hepatoma HepG2,SMMC-7721 cells were transfected with pEGFP-LC3 plasmid to test expression of LC3 by Confocal laser scanning microscope.Experiment 2:The role of autophagy induced by melittin on cell death in HCC cells.1、MTT was used to detect the proliferation inhibition of hepatoma HepG2,SMMC-7721 cells in autophagy inhibitor,autophagy activator,combining with Mel5μg/mL for 24h respectively.Annexin V-FIFC/PI double staining was used to detect the apoptosis rate of hepatoma HepG2,SMMC-7721 cells in autophagy inhibitor,autophagy activator,combining with Mel 5μg/mL for 24h respectively.Trypan blue staining was used to detect the cell death of hepatoma HepG2,SMMC-7721 cells in autophagy inhibitor,autophagy activator,combining with Mel 5μg/mL for 24h respectively.2、After Hepatoma HepG2,SMMC-7721 cells in autophagy inhibitor and combining with Mel 5μg/mL respectively,Western Blot was used to test protein expression of Beclin1 complexes and apoptotic pathways.Experiment 3:The effect of melittin on HCC cells growth in vivo.1、We used BALB/C mice to establish human liver cancer nude mice model treated with inoculating SMMC-7721 cells at right lower extremity subcutaneously.2、Nude mice were divided into six groups randomly:negative control group(0.9%sodium chloride injection),CQ(Chloroquine)group[40mg/(kg·d)],Mel low-dose treatment group[50μg/(kg·d)],Mel high-dose treatment group[100μg/(kg·d)],Mel low-dose treatment+CQ group,Mel high-dose treatment+CQ group.Mel groups were treated by caudal vein injection and CQ groups were treated by intraperitoneal injection with 0.2mL/d for one week.The negative control group was injected with the same volume of normal saline by caudal vein and abdominal cavity.Tumors were measured every two days about long diameter(a)and minor axis(b),according to the formula V=ab~2/2.Once the treatment was over,we killed mice and peelled tumors.At last,we calculated the inhibition rate.ResultsExperiment 1:Melittin inhibits HCC cells proliferation and induces autophagy.1、At the same time,with the increase of Mel concentration,the inhibition rate of HepG2,SMMC-7721 cells proliferation was gradually increased.Compared with the control group,except Mel 1μg/mL for 24,48 h,other concentrations were significant(P<0.05);In the same concentration,with the extension of Mel,the inhibition rate of HepG2,SMMC-7721 cells proliferation was gradually increased.Compared with the control group,except Mel 1,2μg/mL,others were statistically significant(P<0.05).Inverted phase contrast microscope was applied to observe the morphological change of hepatoma HepG2,SMMC-7721 cells in different concentration of Mel for 24h.Microscopically,compared with the control group,cells in Mel became in bad state,edge enhanced and irregular.With the drug concentration increasing,cells floated losing their characteristics.After HepG2,SMMC-7721 cells in different concentration of Mel for 24h,with the drug concentration rising,the proportion of apoptotic cells increased gradually,necrotic cell percentage became increased significantly(P<0.05).2、After HepG2,SMMC-7721 cells in different concentration of Mel for 24 h,Western Blot results showed:with the drug concentration increasing,the expression of LC3Ⅰ/Ⅱ,Beclin 1 gradually increased,while the expression of p62 decreased gradually;After HepG2,SMMC-7721 cells in Mel 5μg/mL for different times,with the extension of Mel,the expression of LC3Ⅰ/Ⅱ,Beclin 1 gradually increased,the expression of p62decreased gradually(P<0.05).Transmission electron microscopy showed:compared with the control group,the phenomenon of“vacuole”in cytoplasm was observed.After HepG2,SMMC-7721 cells in Mel 5μg/mL for 24 h,Bright fluorescence of Beclin 1 in Mel treatment group could be observed and the expression increased significantly compared with the control group.When hepatoma HepG2,SMMC-7721 cells transfected with pEGFP-LC3 plasmid were treated by Mel 5μg/mL for 24 h,confocal laser scanning microscope showed:the LC3 dots in Mel treatment group was higher than the control one significantly(P<0.05).Experiment 2:The role of autophagy induced by melittin on cell death in HCC cells.1、After HepG2,SMMC-7721 cells in Mel 5μg/mL for 24 h,the inhibition rate of Mel5μg/mL combined with autophagy inhibitorwas higher than other ones significantly(P<0.05),the apoptosis rate of Mel 5μg/mL combined with autophagy inhibitor was higher than other ones significantly(P<0.05),the cell survival rate of Mel 5μg/mL combined with autophagy inhibitor was lower than other ones significantly(P<0.05).2、After HepG2,SMMC-7721 cells in Mel 5μg/mL for 24 h,Western Blot results showed:compared with the control group,the protein of Beclin 1 expression is increased while Bcl-2 expression is decreased(P<0.05).While Mel combining with CQ,the protein of both beclin 1 and Bcl-2 expressin are decreased,and the cytc、cleaved-caspase 9、cleaved caspase 3 are increased,compared with the Mel group(P<0.05).Experiment 3:The effect of melittin on HCC cells growth in vivo.1、97.5%of the nude mice were successfully established in tumor-bearing model.After a week,the tumor size was smaller in Mel combining with CQ groups than in Mel groups and in control group at the same dose(P<0.05).2、Transmission electron microscopy showed:compared with the Mel groups,the phenomenon of“vacuole”in cytoplasm was rare observed in Mel combining with CQ groups.3、TUNEL detection showed that more tumor cells were detected as apoptosis in Mel combining with CQ groups than Mel groups(P<0.05).Conclusion1、Mel could inhibit the proliferation,induce the apoptosis and necrocytosis of HepG2,SMMC-7721 cells,and it could induce autophagy in HCC cells at the same time.2、Inhibitted autophagy could enhance the Mel effect of inhibiting proliferation,inducing apoptosis and necrocytosis in HepG2,SMMC-7721 cells.3、The mechanim of inhibitting autophagy enhanced the Mel effect on inducing apoptosis may related to inactivating Beclin 1 complexes,reducing the express of Bcl-2and promote the release of cyto-c in turn activates the mitochondrial pathway of apoptosis.